Extracellular RNAs (exRNAs) are present in human serum. It remains unclear to what extent these circulating exRNAs may reflect human physiologic and disease states. Here, we developed SILVER-seq (Small Input Liquid Volume Extracellular RNA Sequencing) to efficiently sequence both integral and fragmented exRNAs from a small droplet (5 μL to 7 μL) of liquid biopsy. We calibrated SILVER-seq in reference to other RNA sequencing methods based on milliliters of input serum and quantified droplet-to-droplet and donor-to-donor variations. We carried out SILVER-seq on more than 150 serum droplets from male and female donors ranging from 18 y to 48 y of age. SILVER-seq detected exRNAs from more than a quarter of the human genes, including small RNAs and fragments of mRNAs and long noncoding RNAs (lncRNAs). The detected exRNAs included those derived from genes with tissue (e.g., brain)-specific expression. The exRNA expression levels separated the male and female samples and were correlated with chronological age. Noncancer and breast cancer donors exhibited pronounced differences, whereas donors with or without cancer recurrence exhibited moderate differences in exRNA expression patterns. Even without using differentially expressed exRNAs as features, nearly all cancer and noncancer samples and a large portion of the recurrence and nonrecurrence samples could be correctly classified by exRNA expression values. These data suggest the potential of using exRNAs in a single droplet of serum for liquid biopsy-based diagnostics.
Author(s): Chen, Chien-Ju | Advisor(s): Jokerst, Jesse V | Abstract: The survival rate of patients with ovarian cancer is over 90% in Stages I and II but detection strategies based on CA125 testing are ineffective. Hence, this screening approach not only fails to decrease ovarian cancer mortality but also causes significant harm including major surgery in cancer free women. Fortunately, the metrics of ultrasound-based techniques are encouraging—a recent large scale trial of over 200,000 women indicated that the sensitivity and specificity were 89.4% and 99.8%, respectively, when combined with CA125 tests. However, the positive predictive value of ovarian cancer was only 43.3%. Therefore, we hypothesize that using photoacoustic ultrasoundcombined with lipid-polymer hybrid nanoparticles can achieve advances in specificity and sensitivity that are need in population-wide studies. In our study, we used poly-lacticco- glutamic acid (PLGA), lecithin, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)]-5000 (DSPE-PEG-folate) to fabricate hybrid nanoparticles. The photoacoustic imaging agents DiR or IR-1061 were encapsulated in PLGA hydrophobic core. The folate-functionalized nanoparticles can actively target HeLa cells which overexpress folate receptors on their cell membrane. The main hypothesis is that the folate-functionalized nanoparticles could produce highercontrast between ovarian cancer tissues and normal tissues than nanoparticles without folate modification. Also, we proved our IR-1061-loaded nanoparticles had a pH-responsive release profile. We can even use photoacoustic imaging to track the releasingIR-1061 in PBS at different pH values. In the future, we attempt to conjugate IR-1061 molecules with anti-cancer drugs to endow our nanoparticles theranostic capability. Our long-term goal is to achieve in vivo photoacoustic imaging and theranostic treatment of ovarian cancer patients.
Abstract In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.
Periodontal probing is a useful diagnostic tool to estimate the periodontal pocket depth and assess the status of periodontal disease, but is limited by systematic and random errors. Here, we used photoacoustic imaging in tandem with a food grade cuttlefish ink contrast agent to specifically measure pocket depths in swine models (n=27 teeth) and then compared this to Williams probe. Photoacoustic imaging used a Vevo LAZR imaging system (Visualsonics) at 40 MHz. Spectral data was collected at both 680 and 800 nm to discriminate between the photoacoustic signal from stains and contrast agent. The pocket depths were measured on the sagittal view of the 3D images as well as with a Williams probe before photoacoustic imaging. The Bland-Altman plots show that 97% of our samples fell within ± 1.96 standard deviations of the differences between the depths measured by photoacoustic imaging and the probe (95% confidence interval) at mesial, lingual and buccal, and distal locations. Small bias values of -0.04, +0.17, and -0.2 mm were identified at mesial, lingual and buccal, and distal locations, respectively; the 95% confidence intervals are plotted as well and all are < 1.0 mm. The photoacoustic imaging approach also offered 0.01 mm precision and could cover the entire pocket versus the probe-based approach that is limited to only a few sites.
Abstract Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1 , a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.
We thank Drs. Hartl and Gao for pointing out the difference in read lengths between the cancer and normal samples (1). Indeed, Illumina discontinued their 50-base pair (bp) sequencing kits after we finished sequencing the cancer samples, forcing us to switch to 75-bp sequencing kits on the normal samples. The different sequencing kits did incur small changes of sequence alignment metrics. While acknowledging the possibility that this may bias the assessment of cancer vs. normal discrimination performance of our method, we believe such a possibility is substantially ameliorated by the following considerations. First, a recent study reported that “with the exception of 25 bp reads, there is little difference for the detection of … [↵][1]1To whom correspondence may be addressed. Email: shuchien{at}ucsd.edu, hisu{at}ucsd.edu, or szhong{at}ucsd.edu. [1]: #xref-corresp-1-1
Abstract The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells. These maps reveal the chromosomal domains demarcated by locally transcribed RNA, hereafter termed RNA-defined chromosomal domains. Further, the spreading of caRNA is constrained by the boundaries of topologically associating domains (TADs), demonstrating the role of the 3D genome structure in modulating the spatial distribution of RNA. Conversely, stopping transcription or acute depletion of RNA induces thousands of chromatin loops genome-wide. Activation or suppression of the transcription of specific genes suppresses or creates chromatin loops straddling these genes. Deletion of a specific caRNA-producing genomic sequence promotes chromatin loops that straddle the interchromosomal target sequences of this caRNA. These data suggest a feedback loop where the 3D genome modulates the spatial distribution of RNA, which in turn affects the dynamic 3D genome organization.
This protocol accompanies the manuscript Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization (https://doi.org/10.1101/2023.08.16.553610). It protocol outlines the steps for the enzymatic amplification and preparation of padlock probes from an oligo pool. This strategy has a lower upfront cost compared to individual synthesis of probes and thus permits the use of tens of thousands of padlock probes per probe set. Additionally, the oligo pool serves as an unlimited source of padlock probes, as it can be expanded by PCR amplification upon need.
